Chromosomal abnormalities detected by chromosomal microarray analysis and pregnancy outcomes of 4211 fetuses with high-risk prenatal indications

With the gradual liberalization of the three-child policy and the development of assisted reproductive technology in China, the number of women with high-risk pregnancies is gradually increasing. In this study, 4211 fetuses who underwent chromosomal microarray analysis (CMA) with high-risk prenatal indications were analysed. The results showed that the overall prenatal detection rate of CMA was 11.4% (480/4211), with detection rates of 5.82% (245/4211) for abnormal chromosome numbers and 5.58% (235/4211) for copy number variants. Additionally, the detection rates of clinically significant copy number variants were 3.78% (159/4211) and 1.8% (76/4211) for variants of uncertain significance. The detection rates of fetal chromosomal abnormalities were 6.42% (30/467) for pregnant women with advanced maternal age (AMA), 6.01% (50/832) for high-risk maternal serum screening (MSS) results, 39.09% (224/573) with abnormal non-invasive prenatal testing (NIPT) results, 9.21% (127/1379) with abnormal ultrasound results, and 5.1% (49/960) for other indications. Follow-up results were available for 4211 patients, including 3677 (3677/4211, 87.32%) whose infants were normal after birth, 462 (462/4211, 10.97%) who terminated their pregnancy, 51 (51/4211, 1.21%) whose infants were abnormal after birth, and 21 (21/4211, 0.50%) who refused follow-up. The results of this study demonstrate significant variation in the diagnostic rate of chromosomal microarray analysis across different indications, providing valuable guidance for clinicians to assess the applicability of CMA technology in prenatal diagnosis.


AMA only
The pregnant women in this cohort were aged over 35 years and exhibited no other indications, with an average age of 39.3 years (35-53 years old).There were 30 (30/467, 6.42%) patients whose fetuses had chromosomal abnormalities detected by CMA, including 10 with aneuploidy (10/467, 2.14%) and 20 (20/467, 4.28%) with CNVs.A total of 467 patients of AMA were categorized into three subgroups according to maternal age (see Table 2), and the relationship between maternal age and the chromosomal abnormality rate was analysed.The

Abnormal MSS results
The center screened the serum of pregnant women in the second trimester by detecting the levels of AFP and free β-HCG.CMA analysis revealed that 50 of the 832 patients had abnormal MSS results (50/832, 6.00%), including 16 with aneuploidy (16/832, 1.92%) and 34 with CNVs (34/832, 4.08%).The 832 patients with abnormal MSS results were categorized into three subgroups according to risk type: 721 patients with a high risk of trisomy 21 syndrome (721/832, 86.66%), 28 patients with a high risk of trisomy 18 syndrome (28/832, 3.36%) and 83 patients with a moderate risk of trisomy 21 syndrome (83/832, 9.98%).Among them, 6 patients were diagnosed with trisomy 21 syndrome and included in the high-risk group for trisomy 21.There were 5 patients with trisomy 18 syndrome, 4 of whom had a high risk of trisomy 21 syndrome and 1 who had a high risk of trisomy 18 syndrome (see Table 3).

Abnormal NIPT results
A total of 573 patients with abnormal NIPT results, including chromosomal aneuploidy and CNVs detected by NIPT, were evaluated using CMA.4).Moreover, the detection rate of abnormal chr21 was significantly higher than that of other autosomal abnormalities (73.91% vs. 22.98%, p < 0.001).The detection rate of sex chromosomal abnormalities was also significantly higher than that of other autosomal abnormalities (48.04% vs. 22.98%, p < 0.001).

Detection of ROH by the CMA platform
CMA was used to identify 11 fetuses with ROH (see Schedule 2), including 1 with a whole X chromosome, 1 with a whole chromosome 4, 2 with a long arm fragment on chromosome 15, 1 with a short arm fragment on chromosome 16, 2 with a partial fragment on chromosome 8, 1 with a short arm fragment on chromosome 5, 1 with a long arm fragment on chromosome 12 and 8, 1 with a long arm fragment on chromosome 10, and 1 case with a long fragment on chromosome 2, 18 and 13.
In the group with abnormal NIPT results, chromosome aneuploidy and CNVs were detected across all 24 chromosomes through NIPT.Due to the use of different NIPT platforms, which employ varying sequence read depths and algorithms provided by multiple providers, accurate assessment of positive predictive values was not feasible.The positive predictive values for the detection of other autosomes were consistently lower than those observed for abnormal 21, abnormal 18, abnormal 13, and sex chromosomes in the general population, irrespective of the platform employed.Therefore, the screening efficacy of NIPT for detecting other chromosome aneuploidies and CNVs remains limited.Due to the technical limitations of NIPT, the detected material is derived from placental cell-free DNA, leading to false-positive results.Among the 573 patients with abnormal NIPT results in this study, 349 had normal results after prenatal diagnosis.Possible factors included confined placental mosaicism, early disappearance or cessation of the development of one of the twins, and maternal chromosomal abnormalities or maternal diseases.
Ultrasound soft markers are frequently observed in fetuses with chromosomal abnormalities, however, the indications to perform CMA in fetuses with soft markers are not standardized.Several typical cases were identified in this study.Two fetuses with 6p25 deletion syndrome were detected, and all clinical indications suggested thickened nuchal translucency.Three fetuses with fetal renal cysts and diabetes syndrome showed enhanced renal echo during prenatal ultrasound examination.These findings are consistent with the published literature showing that the most common pathogenic CNV in fetuses with renal and urinary tract abnormalities is 17q12 microdeletion, which leads to renal cysts and diabetic syndrome 23 .The presence of clinically significant CNVs may be observed when prenatal ultrasound indicates thickened nuchal translucency or enhanced renal echo.Fetuses with ultrasound soft marker abnormalities exhibited varying incidence rates of CNVs (P/LP).Our research revealed a small but not statistically significant increase in the likelihood of clinically relevant CNVs in fetuses with one or more ultrasound soft markers (3.1% vs. 4.29%, p = 0.853), consistent with previous investigations 24 .These findings suggested that CMA should be performed in pregnant women when two or more ultrasound soft markers are detected by ultrasound.
Among the subgroup of fetuses with ultrasound structural abnormalities, the detection rate of CNVs (P/ LP) was the highest in the group of fetuses with structural anomalies combined with soft marker abnormalities (8.2%), followed by those with multiple system structural abnormalities (7.41%).The detection rate of CNVs (P/LP) in fetuses with a single-system malformation is similar to that previously reported (3.1-7.9%) 25 , with skeletal (9.52%), genitourinary (2.2%), central nervous (9.09%) and cardiovascular system abnormalities (3.43%) being the most commonly associated with CNVs (P/LP).These results showed that CMA should be recommended when fetal ultrasound reveals multiple structural abnormalities, especially in skeletal, genitourinary, central nervous, and cardiovascular system abnormalities.In this study, Two fetuses with Emanuel syndrome were identified, and CMA results showed 11q23.3q25region duplication and 22q11.1q11.21region duplication.www.nature.com/scientificreports/Prenatal ultrasound revealed a single umbilical cord and underdeveloped cerebellar vermis in one fetus, while the other exhibited a posterior fossa anomaly.These are the most common defects of ES and might be diagnosed in early pregnancy 26 .The clinical phenotype of the 22q11.2proximal deletion exhibits significant heterogeneity, primarily including heart defects, palatal abnormalities, developmental retardation, and immunodeficiency.In this study, Two fetuses with a 22q11.2proximal deletion had cardiac defects: one exhibited right foot varus and left ventricular punctate strong echoes, and the other presented with a right aortic arch.22q11.2deletion is frequently observed in fetuses with heart defects diagnosed prenatally 27 .Therefore, CMA can be used as a detection method for prenatal echocardiography abnormalities.The phenotype resulting from a susceptibility CNV is unpredictable due to incomplete penetrance and variable expressivity 28,29 .These features have not been systematically described although the clinical phenotypic features associated with postnatal recurrent microdeletion/duplication syndromes are well-defined, these features have not been systematically described in prenatal cases due to the limitations of prenatal identification.In our study, the most common recurrent microdeletion/duplication syndromes were 1q21.1 deletion syndrome (10 fetuses) and 15q11.2deletion syndrome (10 fetuses).22q11.2duplication syndrome (9 16p13.11duplication syndrome (9 fetuses), proximal 16p12.2microdeletion syndrome (7 fetuses), 16p11.2microdeletion syndrome (4 fetuses), and 16p11.2duplication syndrome (3 fetuses), 16p13.11deletion syndrome (2 fetuses) were also common in this study.Among these fetuses, only 7 (7/54, 12.2%) exhibited structural abnormalities and all pregnancies were terminated.Moreover, we observed that the proportion of normal fetuses after birth is greater in recurrent microdeletion/duplication syndromes with penetrance of less than 10%.For example, approximately 90% of fetuses with 15q11.2deletion syndrome and 16p13.11duplication syndrome have a normal outcome.Therefore, we recommend not informing couples about CNVs classified as loci with penetrance of less than 10%.These potential neurodevelopmental sites may have some structural abnormalities and may not have obvious clinical indications during pregnancy, which will increase parents' anxiety about fetal development.The precise ultimate phenotype remains unknown, posing significant challenges for genetic counseling.
The single nucleotide polymorphism array (SNP array) technology of CMA can detect not only CNVs, but also ROH, uniparental disomy (UPD), and low-level mosaics 30 .As chromosomes 6,7,11,14,15 and 20 are known to be associated with parental-specific expression genes, further testing is necessary to clarify the diagnosis and distinguish between ROH and UPD.In clinical practice, child-parent trio analysis through CMA is often required 31 .This study reported two cases of fragmented ROH involving the long arm of chromosome 15.Due to AMA and the absence of future childbearing plans, both couples abandoned further diagnostic tests and terminated their pregnancies, so we could not determine the pathogenicity of fragmented ROH involving the long arm of chromosome 15.A case of fragmented ROH on the short arm of chromosome 5 was reported.The karyotype of this fetus was 46, XY, del(5)(p13) [32]/46, XY [70], which led to mosaic loss in the critical region causing Cri du chat syndrome.Consequently, the couple decided to induce labor.When a critical region causing microdeletion syndrome exhibits ROH, it is imperative to complement this region with karyotyping analysis to ascertain its pathogenicity.The presence of multiple large regions of ROH on various chromosomes may indicate a potential consanguineous relationship between the tested individual's parents, either close or distant 32 .In this study, one fetus was found to have multiple large regions of ROH on various chromosomes due to consanguineous marriage between the parents.The couple ultimately decided to terminate the pregnancy.The presence of ROH on the whole of chromosome 4 was identified in our study.In the advanced stages of gestation, intrauterine growth restriction led to fetal demise within the uterus.This finding was consistent with previous studies that fetuses with ROH frequently exhibited the most prevalent prenatal manifestation of intrauterine growth restriction 33 .For 6 fetuses involving other chromosome fragmentary ROH regions, all six couples opted to continue their pregnancies, and subsequent assessments confirmed normal fetal development postpartum.Due to the complex pathogenesis of ROH, encompassing gene imprinting effects, homozygous recessive gene mutations, and low-level chromosomal mosaics, a comprehensive assessment of prognosis should be conducted by combining ultrasound findings, family verification, whole exome sequencing, and other relevant factors.
The pregnancy outcomes of all women were evaluated in this study.Although the majority of women with fetuses diagnosed with aneuploidy or CNV(P/LP) opted for pregnancy termination, 70 women whose fetuses with aneuploidy or CNVs (P/LP) decided to continue their pregnancy, and subsequent follow-up revealed that 65 of the newborns exhibited normal phenotypes.Notably, among these patients, 50% had fetuses with sex chromosome aneuploidy.The results of our investigation indicated that an increasing number of people are accepting of children with sex chromosome aneuploidy.In this study, 76 fetuses with CNVs (VUS) were found, for a detection rate of 1.85%.After follow-up, a total of 45 fetuses were delivered, and among these fetuses, 44 exhibited normal postnatal outcomes.This finding indicates that fetuses with CNVs (VUS) are most likely to have a good pregnancy outcome.Furthermore, reporting VUS in prenatal diagnosis may present challenges for genetic counseling, place pressure on pregnant women and their families, and lead to excessive termination of pregnancy.Therefore, in subsequent stages, further case accumulation and long-term follow-up are needed to comprehensively evaluate the prognosis.This study has significant implications for the development of future genetic counseling guidelines.
Due to the limited availability of detailed genotype-phenotype information for some fetuses, this retrospective study is limited in terms of data acquisition.For instance, some pregnant women have poor compliance or refuse to undergo testing due to financial constraints, while others may experience information loss during the referral process, resulting in the potential omission of high-risk indications for certain fetuses and ultimately introducing bias into the study findings.Furthermore, incomplete clinical examinations in fetuses with a young gestational age may lead to insufficient descriptions of certain clinical phenotypes.Ultimately, the absence of parental data presents a significant challenge in assessing the pathogenicity of certain CNVs, thereby complicating our clinical genetic counseling efforts.
The CMA test is applicable for all prenatal clinical indications because it enhances the detection of clinically significant CNVs.Clinicians should duly apprise pregnant women about the potential risk of undetectable CNVs through biochemical screening or most existing NIPT platforms, and emphasize the value of CMA in prenatal diagnosis for informed decision-making.

Patients and clinical indications
All patients who underwent invasive prenatal diagnosis by CMA at Linyi Women and Children's Hospital between 2016 and 2022 in Shandong, China.Clinical samples (chorionic villi and amniotic fluid) were obtained by ultrasound-guided abdominal chorionic villus sampling (CVS) and amniocentesis.After receiving detailed genetic counseling before testing, each participant signed a written informed consent form.This study was approved by the Ethics Committee of Linyi Maternal and Child Hospital (No. KYL-YXLL-2022017).The research was conducted in accordance with the relevant guidelines and clinical norms.

Statistical analysis
SPSS 21.0 (Chicago, USA) was used for the statistical analysis.Classified variables are expressed as numbers and percentages, and the chi-square test was used for comparisons.A two-sided p-value < 0.05 was considered statistically significant.Linear regression models with gamma values were used to assess the changes in abnormal rates in different age groups.

Table 1 .
CMA results of 4211 samples with different indications for prenatal diagnosis.***The significance is at the p < 0.001 level.

Table 2 .
CMA results of 467 samples with only AMA.

Table 3 .
CMA results of 832 samples with abnormal MSS.

Table 4 .
CMA results of 573 samples with abnormal NIPT.

Table 6 .
Follow-up results of 4211 pregnant women tested for CMA.